Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1997-11-13
2002-01-15
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S004000, C800S015000, C800S016000, C800S017000, C800S018000, C435S320100
Reexamination Certificate
active
06339183
ABSTRACT:
BACKGROUND OF THE INVENTION
Methods of producing biologically active molecules by transfer of recombinant genes into cell in culture and into live animals have been developed. For example, DNA molecules have been introduced into cultured cells by calcium phosphate precipitation or electroporations. Graham and Van der Ebb
Virology
1973, 52, 456-467; Perucho et al.
Cell
1980, 22, 9-17; Chu et al.
Nucleic Acids Research
1987, 15, 1311-1326; and Bishop and Smith
Molecular Biology Medicine
1989, 6, 283-298. DNA molecules have also been introduced into the nucleus of cells in culture by direct microinjection. Gordon et al.
Proc. Natl Acad. Sci. USA
1980, 77, 7380-7384; Gordon and Ruttel
Methods in Enzymology
1983, 101, 411-433; and U.S. Pat. No. 4,873,191.
However, there are two major problems of producing biologically active molecules such as protein products on a commercially viable scale via these methods. First, bacterial expression systems frequently fail to modify the proteins properly, i.e., by glycosylation, etc. Second, the subsequent isolation of gene products from the expression systems can be extremely difficult. In bacteria, yeast, and baculovirus systems the expressed proteins are most often purified from insoluble intracellular compartments. Secreted proteins in yeast require specialized protease-deficient strains coupled with appropriate vectors with secretion signals.
Retroviral vectors have also been used to introduce DNA molecules into the genome of animals. Jaenisch et al.
Cell
1981, 24, 519; Soriano et al.
Science
1986, 234, 1409-1413; and Stewart et al.
Embo. J.
1987, 6, 383-388. Recombinant genes have been introduced into primary cultures of bone marrow, skin, fibroblasts, or hepatic or pancreatic cells and then transplanted into live animals. There has also been success in using mammary gland-specific promoters to drive the expression of foreign proteins in these secretory glands, ultimately leading to their secretion in the resultant milk. This method has been used commercially to express human growth hormone in cows and sheep. WO 94/05782. The copious volumes of milk produced by cows and sheep make this procedure attractive. However, this method suffers from several potential drawbacks: one being that the expressed protein even at relatively high levels must be purified away from a large amount of milk proteins such as caseins, immunoglobins, lactoferrins which may also entrap the desired valuable product; another being that certain protein products may be insoluble in the calcium-rich environment of milk fluid; and another being that this method requires the use of pregnant animals which are expensive and time consuming to produce.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a vector comprising a promoter construct capable of directing urothelial gene expression of a heterologous gene encoding a selected biologically active molecule linked thereto. The vectors of the present invention are useful in directing the expression of the heterologous gene in urothelial cells transfected with the vector which then secrete the encoded gene product into the urine for isolation, thus transforming the bladder into a bioreactor.
Accordingly, another object of the present invention is to provide a method of producing a selected biologically active molecule in urine of an animal wherein urothelial cells in the animal are transfected with the vector so that the heterologous gene of the DNA sequence is expressed and the selected biologically active molecule is recovered in urine produced by the animal.
Another object of the present invention is to provide nonhuman transgenic animals produced using this vector.
Another object of the present invention is to provide animal models for human bladder cancer.
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Browdy And Neimark
Crouch Deborah
New York University
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